Methods of detecting specific cell lysis

ABSTRACT

The present invention provides methods of detecting specific lysis of a cell by a lytic agent. The methods generally involve contacting a labeled target cell with a lytic agent; and detecting fluorescence in the target cell. The target cells are labeled with two fluorescent labels: a first fluorescent label that labels the plasma membrane; and a second fluorescent label that labels the cytosol. Release of the cytosolic label from the target cell indicates that the target cell has been lysed. The invention further provides methods of detecting the presence in a sample of a cell that specifically lyses a target cell. The invention further provides methods of detecting the presence in a sample of an antibody that specifically lyses a target cell. The methods are useful in a variety of applications.

CROSS-REFERENCE

This application claims benefit of priority of U.S. Provisional PatentApplication Ser. No. 60/282,258, filed Apr. 5, 2001, which applicationis incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. government may have certain rights in this invention, pursuantto grant no. RO1-A146254 awarded by the National Institutes of Health.

FIELD OF THE INVENTION

The present invention is in the field of cellular biology, and inparticular relates to cell lysis.

BACKGROUND OF THE INVENTION

Specific lysis of target cells is a feature of a number of normalphysiological processes. For example, CD8⁺ cytotoxic T lymphocytes (CTL)are central to the adaptive immune response against viruses, protozoa,intracellular bacteria, and in the rejection of allogeneic grafts. CTLrecognize and kill target cells expressing antigen-derived peptidespresented by class I major histocompatibility complex (MHC). In vivo,antigen specific CD8⁺ T cells also exert effector activity throughrelease of soluble cytokines and chemokines, which either signal othereffector cells or have direct antiviral activity. Other cells have lyticactivity and participate in the elimination of infected cells or, insome circumstances, self derived cells as in autoimmune diseases. Theselytic cells include CD4⁺ T lymphocytes with lytic effects, NaturalKiller (NK) cells, Natural Killer T (NKT) cells and lymphokine activatedkiller (LAK) cells.

A number of cells that have cytotoxic potential express membranereceptors for the Fc region of an antibody molecule. When antibody isspecifically bound to a target cell, receptor-bearing lytic cells bindto the target cell-bound antibody Fc region, and subsequently causelysis of the target cell. Although the lytic cells are not themselvesspecific, the specificity of antibody directs them to the specifictarget cells, in a reaction known as antibody-dependent cell-mediatedcytotoxicity (ADCC). Among the cells that can mediate ADCC are naturalkiller (NK) cell, macrophages, monocytes, neutrophils, and eosinophils.Antibodies can also be used to redirect effector lytic cells towardtarget cells.

Specific lysis of target cells is also a feature of certain aberrantphysiological processes. Self-reactive T lymphocytes and antibodies areinvolved in the destruction of cells in autoimmune disorders such asinsulin-dependent (Type 1) diabetes mellitus and autoimmune hemolyticanemia. Graft-versus-host disease (GVDH) develops when immunocompetentlymphocytes are injected into an allogeneic recipient whose immunesystem is compromised, e.g., a patient who has had radiation exposure orwho has leukemia, immunodeficiency disease, or an autoimmune anemia andwho is a recipient of an allogeneic bone marrow transplant. The graftedlymphocytes attack the host, whose immunocompromised state prevents anadequate immune response against the graft.

The cytolytic activity of CD8⁺ T cells has been commonly determined bythe ⁵¹Chromium (⁵¹ Cr) release assay. The standard ⁵¹Cr release assayhas a number of disadvantages that include high spontaneous release,influence of ⁵¹Cr upon the effector cell population, problems withlabeling certain cell types, low sensitivity, and health risksassociated with gamma irradiation. Assays such as the modified enzymelinked immuno-assay (ELISPOT), which detects the secretion of cytokinesfollowing antigenic stimulation, and the use of tetrameric MHC class Icomplexes have afforded greater sensitivity in the detection of antigenspecific CD8⁺ T cells.

Non-radioactive alternatives to the standard cytotoxic assays haveincluded detection of released intracellular enzymes, calorimetricassays or detailed preparation of reporter cell lines. In addition,assays that employ the use of flow cytometry by the detection offluorescent dyes for either lymphocyte-target conjugate formation orcytolytic activity have been described. Flow cytometric cytotoxicityassays generally involve the measurement of a fluorochrome releasedfrom, or remaining in, pre-labeled effector cells or targets,simultaneously or exclusively. Ideally, these labels should not changethe morphology or function of the labeled cells. However, some of thefluorochromes that have been proposed label target cells poorly, requirecomplex manipulations of light scatter properties to discriminate theviable cell population or have a higher spontaneous release compared tostandard ⁵¹Cr release. In addition, fluorometric techniques which relyon the incorporation of a nucleic acid stain such as propidium iodide,do not account for active phagocytic cells that can take up dead cellsin vitro. As a consequence, none of these proposed assays have gainedacceptance, or replaced the ⁵¹Cr release assay, which remains inwidespread use.

To improve the study of in vitro cytolytic function of lytic agents suchas cytolytic cells and antibodies, there is a need for a reliable assaythat analyzes subpopulations, eliminates the requirement for potentiallyhazardous radioactive isotopes, offers increased sensitivity, utilizesan efficient label that is detected easily with low spontaneous releaseand is reproducible. The present invention addresses this need.

Literature

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SUMMARY OF THE INVENTION

The present invention provides methods of detecting specific lysis of acell by a lytic agent. The methods generally involve contacting alabeled target cell with a lytic agent; and detecting fluorescence inthe target cell. The target cells are labeled with two fluorescentlabels: a fluorescent label that labels the plasma membrane; and afluorescent label that labels the cytosol. Release of the cytosoliclabel from the target cell indicates that the target cell has beenlysed. The invention further provides methods of detecting the presencein a sample of a cell that specifically lyses a target cell. Theinvention further provides methods of detecting the presence in a sampleof an antibody that specifically lyses a target cell. The methods areuseful in a variety of applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D depict staining of cells with PKH-26 and CFSE, alone and incombination.

FIG. 2 depicts lysis of peptide-pulsed target cells stained with CFSEand PKH-26.

FIG. 3 is a graph depicting spontaneous release of CFSE and ⁵¹Cr fromlabeled cells.

FIG. 4 is a graph depicting peptide-specific lysis of CFSE- or⁵¹Cr-labeled target cells.

FIGS. 5A and B depict FACS plots showing staining of donor BCL with CFSEand either PKH-26 (FIG. 5A) or DiD (FIG. 5B), and analysis of eachstained cell population individually.

FIG. 6 depicts results showing that dual-labeled target cells arereadily distinguishable in mixed populations using flow cytometry.

FIG. 7 depicts the results of contacting unpulsed labeled target cellswith a lytic agent.

FIG. 8 depicts the results of contacting peptide-pulsed labeled targetcells with a lytic agent.

DEFINITIONS

As used herein, the term “specific lysis” refers to lysis of a targetcell based on the expression on its cell surface of a particularprotein, peptide, glycoprotein, glycolipid, or lipoprotein. Specificlysis can be readily distinguished from non-specific lysis through theuse of appropriate controls, e.g., a control cell (e.g., a cell of thesame cell type, or a cell of same cell line) that does not express onits cell surface the same protein, peptide, glycoprotein, glycolipid, orlipoprotein that is the basis for specific lysis of the target cell.

The term “binds specifically,” in the context of antibody binding,refers to high avidity and/or high affinity binding of an antibody to aspecific polypeptide e.g., an epitope of a polypeptide. Antibody bindingto an epitope on a specific polypeptide is preferably stronger thanbinding of the same antibody to any other epitope, particularly thosewhich may be present in molecules in association with, or in the samesample, as the specific polypeptide of interest, e.g., binds morestrongly to a specific epitope than to a different epitope so that byadjusting binding conditions the antibody binds almost exclusively tothe specific epitope and not to any other epitope, and not to any otherpolypeptide which does not comprise the epitope.

Antibodies which bind specifically to a given polypeptide (or epitope)may be capable of binding other polypeptides at a weak, yet detectable,level (e.g., 10% or less of the binding shown to the polypeptide). Suchweak binding, or background binding, is readily discernible from thespecific antibody binding to the compound or polypeptide of interest,e.g. by use of appropriate controls. In general, antibodies which bindto a specific polypeptide with a binding affinity of 10⁻⁷ M or more,generally 10⁻⁸ M or more (e.g., 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, etc.) are saidto bind specifically to the specific polypeptide. In general, anantibody with a binding affinity of 10⁻⁶ M or less is not considered“specific.”

The term “binds specifically” in the context of an antigen-specific CD8⁺T lymphocyte refers to binding of the CD8⁺ T cell to a particularpeptide presented in a class I MHC molecule on a target cell, but notsubstantially to a different peptide presented in a class I MHC moleculeon a target cell. The term “binds specifically” in the context of anantigen-specific CD4⁺ T lymphocyte refers to binding of the CD4⁺ T cellto a particular peptide presented in a class II MHC molecule on a targetcell, but not substantially to a different peptide presented in a classII MHC molecule on a target cell.

The term “fluorescence” is well known in the art. In the context of afluorescent dye, the term refers to a dye that can be excited at onewavelength of light following which it will emit light at anotherwavelength. Excitation generally occurs at a wavelength in the range offrom about 250 to 750 nm. Emitted wavelengths are generally in the rangeof from about 200 nm to about 300 nm, from about 300 nm to about 400 nm,from about 380 nm to about 400 nm, from about 400 nm to about 430 nm,from about 430 nm to about 500 nm, from about 500 nm to about 560 nm,from about 560 nm to about 620 nm, from about 620 nm to about 700 nm,from about 700 nm to about 1.5 μm, from about 1.5 μm to about 20 μm, orfrom about 20 μm to about 1000 μm.

A fluorescent dye that is “distinguishable” from another fluorescent dyeusing standard detection methods and devices (e.g., flow cytometrydevices), refers to the fact that the spectral properties of the twofluorescent dyes being compared are detectably different from oneanother, e.g., the emission of a given fluorescent dye differs from theemission of a second fluorescent dye by at least about 10 nm to about 15nm, from about 15 nm to about 20 nm, from about 20 nm to about 25 nm,from about 25 nm to about 30 nm, from about 30 nm to about 35 nm, fromabout 35 nm to about 40 nm, from about 40 nm to about 45 nm, from about45 nm to about 50 nm, from about 50 nm to about 55 nm, from about 55 nmto about 60 nm, from about 60 nm to about 65 nm, from about 65 nm toabout 70 nm, from about 70 nm to about 75 nm, from about 75 nm to about80 nm, from about 80 nm to about 85 nm, from about 85 nm to about 90 nm,from about 90 nm to about 95 nm, from about 95 nm to about 100 nm, fromabout 100 nm to about 120 nm, from about 120 nm to about 140 nm, fromabout 140 nm to about 160 nm, from about 160 nm to about 180 nm, or fromabout 180 nm to about 200 nm, or more.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents; washed; orenrichment for certain cell population, such as tumor cells, peripheralblood mononuclear cells (PBMC), lymphocytes, and the like. The term“biological sample” encompasses a clinical sample, and also includescells in culture, cell supernatants, tissue samples, organs, bonemarrow, and the like.

As used herein, the terms “determining” and “detecting” encompassqualitative and quantitative detection, and as such includes “measuring”and equivalent terms.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, e.g., causing regression of the disease,e.g., to completely or partially remove symptoms of the disease.

The terms “individual,” “host,” “subject,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines, simians, humans, mammalian farm animals, mammalian sportanimals, and mammalian pets.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “afluorescent dye” includes a plurality of such dyes and reference to “thetarget cell” includes reference to one or more target cells andequivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for detecting and/or measuringspecific lysis of a target cell; and methods for detecting in a samplethe presence of and/or the number of cells having lytic activity towarda target cell. The methods generally involve labeling a target cell withtwo fluorescent labels: a fluorescent label that labels the plasmamembrane; and a fluorescent label that labels the cytosol. The spectralproperties of the cytosol-labeling fluorescent dye and the plasmamembrane-labeling dye are distinguishable from one another usingstandard detection methods and devices. Release of the cytosolic labelfrom the target cell indicates that the target cell has been lysed. Theinvention is useful in a variety of applications, e.g., in methods todetermine tissue compatibility; in methods to determine the presenceand/or extent of a graft-versus-host reaction; in methods to detect thepresence of cytotoxic T lymphocytes reactive with self cell, with tumorcells, or with pathogen-infected cells; in methods to detect thepresence of autoantibody reactive with a self target cell; and inmethods of measuring an immune response to an antigen. Such methods areuseful in diagnostic assays, e.g., to assist in making an initialdiagnosis of a disease; in diagnostic assays to evaluate the efficacy ofa treatment protocol; and in vaccine testing and evaluation.

The present invention avoids the disadvantages of methods that useradioactive labels such as ⁵¹Cr, such as problems with labeling certaincell types, low sensitivity, health risks associated with usingradioactivity, and a high level of spontaneous release of label. Theinstant methods allow labeling of virtually any cell type. The labelprovides an easily detectable signal of high sensitivity that can bedetected using simple detection methods. Unlike methods involving ⁵¹Crlabeling, in which populations of cells were analyzed, the instantmethods allow analysis of specific cell lysis on an individual cell. Inaddition, unlike previous assays involving labeling cells with a singlefluorescent dye, the present invention involves labeling target cellswith two fluorescent dyes: one that labels the cytosol, and one thatlabels the plasma membrane. Labeling the target cell with both acytosol-labeling fluorescent dye and a plasma membrane-labelingfluorescent dye is advantageous because the only cells that are labeledare viable cells, thereby avoiding the need for use of a dye to detectnon-viable cells (e.g., dead cells or cells undergoing apoptosis) suchas propidium iodide. Furthermore, in the instant methods, the label isreleased from a lysed cell quickly and uniformly, and lysed cells are nolonger fluorescent.

Methods of Detecting Specific Lysis of a Target Cell

The present invention provides methods of detecting specific lysis of atarget cell. The methods generally involve contacting a labeled targetcell with a lytic agent, and detecting a reduction in fluorescence inthe labeled target cell. Target cells are labeled with two fluorescentlabels: a fluorescent label that labels the plasma membrane (a “plasmamembrane-labeling fluorescent dye”); and a fluorescent label that labelsthe cytosol (a “cytosol-labeling fluorescent dye”). Release of thecytosolic label from the target cell indicates that the target cell hasbeen lysed. Specificity is determined by providing suitable controls. 1many embodiments, the methods are performed in vitro. In otherembodiments, the methods are performed in vivo.

In some embodiments, the invention provides methods that involvecontacting a lytic agent with two or more different types of targetcells, in which each different target cell is labeled in such a way thatallows it to be distinguished from the other target cell(s). Thesemethods are advantageous because, in a single reaction mixture, a singlelytic agent can be contacted with multiple target cells. If one type oftarget cell is lysed and a second type of target cell is not lysed, thetwo types of target cells can be distinguished from one another. It isadvantageous to include multiple (e.g., two or more) different types oftarget cells in a single reaction mixture together with a lytic agentbecause it reduces the amount of time it takes to conduct the assays(i.e., less time is required to assay two or more different target cellssimultaneously than would be required to assay individually); it reducesor eliminates the variability that might occur if each target cell wereindividually contacted with the lytic agent and assayed individually;and provides an internal ‘intra-assay’ control for specific lysis of atarget cell.

An example of this method is the inclusion in a single reaction mixtureof a lytic agent, and, as target cells: (1) a target cell that is beingtested for whether it will or will not be lysed by the lytic agent; and(2) a control target cell that is not expected to by lysed by the lyticagent. In this example, a third control target cell that could beincluded is a positive control target cell, i.e., a target cell that isexpected to be lysed by the lytic agent. Each target cell is labeleddifferently, such that the identity of the lysed and unlysed targetcells are known.

Another example of this method is the inclusion in a single reactionmixture of a lytic agent and, as target cells: (1) a target cell from afirst individual; and (2) a target cell from a second individual. Inthis example, control cells (as described in the preceding paragraph),as well as target cells from additional individuals, can also beincluded. As an example, target cells from multiple individuals who arepotential organ donors are mixed with cells from a prospective organrecipient in a single reaction mixture. In this example, the prospectiverecipient's cells are being tested for lytic toward the potentialdonors' cells. Each potential donor's cells are labeled such that theycan be distinguished from all other donors in the reaction mix. The bestmatch between the donor and recipient is identified by determining whichof the potential donor's cells are not lysed.

In some embodiments, the invention provides methods for detectingspecific cell lysis where at least two different target cells arelabeled with at least two different plasma membrane-labeling fluorescentdyes. These methods generally involve (a) contacting a lytic agent with(i) a first target cell labeled with a first plasma membrane-labelingfluorescent dye and a cytosol-labeling fluorescent dye and (ii) at leasta second target cell labeled with a second plasma membrane-labelingfluorescent dye and the cytosol-labeling fluorescent dye; and (b)determining the amount of fluorescent label remaining in the first andat least the second target cell. The method may further include the stepof relating the plasma membrane-labeling fluorescent dye in the unlysedtarget cell to the identity of the unlysed target cell.

A reduction in the amount of the cytosol-labeling fluorescent dye in atarget cell indicates that the target cell is lysed by the lytic agent.The at least two different plasma membrane-labeling fluorescent dyes aredistinguishable from one another by flow cytometry or other device fordetecting fluorescence. Because the at least two different plasmamembrane-labeling fluorescent dyes are distinguishable from one another,if a first target cell labeled with a first plasma membrane-labelingfluorescent dye is lysed and a second target cell labeled with a secondplasma membrane-labeling fluorescent dye is not lysed, the identities ofthe lysed and the unlysed cell are known. Thus, the different plasmamembrane-labeling dyes serve as a code for the different target cells.

In other embodiments, the invention provides methods for detecting lysiswhere at least two different target cells are labeled with at least twodifferent cytosol-labeling fluorescent dyes. These methods generallyinvolve (a) contacting a lytic agent with (i) a first target celllabeled with a first cytosol-labeling fluorescent dye and a plasmamembrane-labeling fluorescent dye and (ii) at least a second target celllabeled with a second cytosol-labeling fluorescent dye and the plasmamembrane-labeling fluorescent dye; and (b) determining the amount offluorescent label remaining in the first and at least the second targetcell. The method may further include the step of relating thecytosol-labeling fluorescent dye in the unlysed target cell to theidentity of the unlysed target cell.

A reduction in the amount of the cytosol-labeling fluorescent dye in atarget cell indicates that the target cell is lysed by the lytic agent.The at least two different cytosol-labeling fluorescent dyes aredistinguishable from one another by flow cytometry or other device fordetecting fluorescence. Because the at least two differentcytosol-labeling fluorescent dyes are distinguishable from one another,if a first target cell labeled with a first cytosol-labeling fluorescentdye is lysed and a second target cell labeled with a secondcytosol-labeling fluorescent dye is not lysed, the identities of thelysed and the unlysed cell are known. Thus, the differentcytosol-labeling dyes serve as a code for the different target cells.

In further embodiments, the invention provides for methods of detectingspecific cell lysis, wherein a plurality of target cells is contactedwith a lytic agent, and wherein a plurality of plasma membrane-labelingfluorescent dyes and a plurality of cytosol-labeling dyes is used tolabel the target cells. For example, a subset of target cells may belabeled in such a way that distinguishes the subset. An examples of asubset are control target cells, which subset in many embodimentsincludes at least two types of control cell.

These embodiments are useful in that more than one target cell or cellpopulation can be analyzed simultaneously with a single lytic agent.Thus, these methods provide for contacting a plurality of target cellswith a single lytic agent. For example, a test target cell and one ormore different control target cells can be analyzed simultaneously witha single lytic agent. As another example, target cells from more thanone individual can be analyzed simultaneously with a single lytic agent.

In some embodiments, the at least two different target cells include afirst target cell that displays a peptide on its surface that isrecognized by an lytic agent; and a second, control target cell lackingthe peptide and/or a control target cell displaying a different peptidefrom the one displayed by the first target cell such that it is notrecognized and lysed by the lytic agent.

In other embodiments, the at least two different cells include a firsttarget cell from a patient, which first target cell is infected with anintracellular pathogen; and a second, control target cell that is notinfected with the intracellular pathogen and/or a control target cellthat is infected with an intracellular pathogen different from the onethat the first target cell is infected with, such that the controltarget cell is not recognized and lysed by the lytic agent.

In still other embodiments, the at least two different target cellsinclude a first target cell from a first individual; and at least asecond target cell from at least a second individual. Multiple targetcells (including control target cells) can be analyzed simultaneouslywith a single lytic agent.

As one non-limiting example, the target cell sample includes: a controltarget cell, a target cell from a first individual being tested fortissue compatibility, and a target cell from a second individual beingtested for tissue compatibility. In this example, the target cell samplecan further include target cells from additional individuals beingtested for tissue compatibility. The target cell sample could thusinclude target cells from multiple potential tissue/organ donors, eachlabeled with a different plasma membrane dye. The target cell sample,including appropriate controls, is then contacted with a lytic agent,which in this case could be a peripheral blood mononuclear cell samplefrom a prospective tissue/organ recipient, or a sample of CD8⁺ cellsfrom the prospective recipient. Thus, in one assay, the potential donorwho presents the best tissue match with the prospective recipient isidentified.

In many embodiments, the subject methods are performed in vitro, e.g.,target cells are labeled in vitro, and contacted with lytic agent invitro. In other embodiments, the subject methods are performed in vivo.In these embodiments, the cells are labeled in vitro, and subsequentlyintroduced into an animal, e.g., an experimental non-human animal, i.e.,the labeled target cell is contacted with the lytic agent in vivo. Insome of these embodiments, the lytic agent is present in theexperimental animal (e.g., the lytic agent is an endogenous cell orantibody having lytic activity toward the target cell); and in otherembodiments, the lytic agent is introduced into the experimental animal.Where the subject method is performed in vivo, detection of cell lysiscan occur in vitro, e.g., the target cells (and the lytic agent) can beremoved from the animal, and subjected to flow cytometry to detectspecific lysis. Alternatively, an in vivo imaging technique can be usedto detect specific cell lysis in vivo.

Fluorescent Labels

Target cells are labeled with two fluorescent labels: a fluorescentlabel that labels the plasma membrane; and a fluorescent label thatlabels the cytosol. The use of a label for the plasma membranefacilitates analysis of the cells, as it allows discrimination betweencells that have been lysed, and cells that have died due to causesunrelated to specific cell lysis.

Fluorescent dyes that label the plasma membrane and are suitable for usein the instant methods are non-cytotoxic, have no effect on biologicalactivities of the cell, yield intense staining with a high signal:noiseratio, label any cell type, provide for uniform labeling of the plasmamembrane, and are compatible with existing detection equipment andstandard fluorescent filters. Fluorescent labels that label the plasmamembrane include lipid-associated fluorescent labels, including, but notlimited to, PKH-26, PKH-67 and long chain dialkylcarbocyanines such as3,3′-dioctadecyloxacarbocyanine perchlorate (DiO),1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate(DiI), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanineperchlorate (DiD), 4-(4-(dihexadecylamino)styryl)-N-methylpyridiniumiodide (DiA); and1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR).Also of interest are variants of DiO, DiI, DiD, DiA, and DiR, including,but not limited to, variants which have shorter alkyl substituents,e.g., C₁₂, and C₁₆ alkyl substituents (e.g., DiIC₁₂, DiIC₁₆, DiOC₁₆, andthe like; variants which have diunsaturated alkyl substituents, e.g.,Δ^(9,12)-C18 alkyl substituents; variants having phenyl substituentsattached to the indoline rings of DiI; sulfonated derivatives of DiI andDiO; and the like. Many such dyes are commercially available. Sigma (St.Louis, Mo.) and Molecular Probes (Eugene, Oreg.) are examples ofcommercial sources of such dyes.

Fluorescent labels that label the cytosol that are useful in the methodsof the present invention include fluorescent labels that permeate theplasma membrane and bind to intracellular proteins in the cytosol.Suitable fluorescent dyes are those that: (1) are spontaneously releasedfrom the cytosol at a very low rate; label virtually any cell type; arereleased from a lysed cell very rapidly, and in one step; do not alterthe morphological or biological characteristics of the cell; label thecells such that they are readily detectable, e.g., labeled cells arebrightly fluorescent; and are detectable using existing equipment andstandard fluorescent filters.

Fluorescent dyes that label the cytosol that are useful in the methodsof the present invention are those that are spontaneously released froma cell at a very low rate, e.g., less than about 15%, less than about10%, less than about 8%, less than about 5%, or less than about 2%spontaneous release occurs during an incubation in standard cultureconditions (e.g., 37° C., 5% CO₂) of from about 2 hours to about 4hours, from about 4 hours to about 8 hours, from about 8 hours to about12 hours, from about 12 hours to about 24 hours, from about 24 hours toabout 36 hours, or from about 36 hours to about 48 hours.

Cytosol-labeling fluorescent dyes that are useful in the methods of theinvention label virtually any cell, including, but not limited to,animal cells, including mammalian cells; plant cells; yeast cells;fungal cells; protozoal cells; and the like.

Fluorescent, cytosol-labeling dyes that are suitable for use in thepresent invention are released rapidly from a lysed cell, e.g., at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or more, of the fluorescent dye is released from alysed cell in about 10 seconds, in about 5 seconds, in about 2 seconds,in about 1 second, or in about 100 milliseconds, or less, of lysis ofthe cell.

In many embodiments of interest, a cytosol-labeling fluorescent label isan uncharged derivative of fluorescein that is cleaved in the cytosol toproduce a charged form that is highly fluorescent. For example,amine-reactive carboxyfluorescein succinimidyl esters that are cleavedby intracellular esterases to yield highly fluorescent amine-reactivefluorophores are suitable. Suitable cytosol-labeling fluorescent dyesinclude, but are not limited to, 5-(-6)-carboxyfluorescein diacetatesuccinimidyl ester (CFSE); CMTMR (5-(6) (((4-chloromethly) benzoyl)amino) tetramethylrhodamine); CMAC (7-amino-4-chloromethylcoumarin); anda SNARF® fluorescent dye. Such dyes are commercially available, e.g.,from Molecular Probes (Eugene, Oreg.). In a particular embodiment ofinterest, the cytosol-labeling fluorescent dye is CFSE.

If desired, target cells and/or effector cells (i.e., cells having lyticactivity toward a target cell) can further be labeled with additionallabels, including, but not limited to, a detectably labeled antibodyspecific for a particular cell surface marker; a dye for detectingnon-viable cells (e.g., propidium iodide); and the like. Such labelingmay be performed to assist in identifying the target and/or effectorcell populations or to gate out non-viable cells.

Lytic Agents

Lytic agents include any agent that effects, directly or indirectly,specific lysis of a target cell. Lytic agents include, but are notlimited to, antibodies specific for a cell surface marker of a targetcell; “effector cells,” including antigen-specific effector cells andnon-antigen-specific effector cells, including, but not limited to, CD8⁺T lymphocytes; CD4⁺ T lymphocytes; NK cells; NKT cells; LAK cells; andcombinations of specific antibody and non-specific effector cells, e.g.an antibody that is specific for a target cell surface molecule, and aneffector cell that bears a cell surface Fc receptor, including, but notlimited to, NK cells, macrophages, eosinophils, monocytes, andneutrophils.

Target Cells

Target cells are any cells that are specifically lysed by a lytic agent,e.g., a lytic cell or a specific antibody. In many embodiments, a targetcell is lysed by a lytic agent that specifically recognizes and binds toa protein, peptide, glycoprotein, glycolipid, or lipoprotein on thesurface of the target cell.

In many embodiments, target cells display an antigenic peptide on theirsurface in a Class I major histocompatibility complex (MHC) molecule. Inmany of these embodiments, the target cell is recognized and lysed bythe lytic cell in an antigen-specific manner, i.e., the lytic cellrecognizes an antigenic peptide presented by the class I MHC moleculeand lyses the target cell. In these embodiments, the lytic cell isgenerally a CD8⁺ T lymphocyte.

In other embodiments, the target cell displays on its cell surface aprotein, peptide, glycoprotein, glycolipid, or lipoprotein that isspecifically recognized and bound by a lytic agent, such as a specificantibody.

In some embodiments, a target cell is a cell line that is charged with apeptide to which a lytic cell is specific.

Antigenic peptides can be bound to the antigen binding site of the MHCmolecule or to the amino-terminus of either an MHC class I chain or a β₂microglobulin. Binding, or “loading” of the antigenic peptide to the MHCmolecule can be carried out actively or passively. The antigenic peptidecan be covalently bound to the chimeric protein. Optionally, a peptidetether can be used to link an antigenic peptide to β₂ microglobulin.Crystallographic analyses of multiple class I MHC molecules indicatethat the amino terminus of β₂ microglobulin is very close, approximately20.5 Å away, from the carboxyl terminus of the antigenic peptideresident in the MHC peptide binding groove. Thus, using a relativelyshort linker sequence, approximately 13 amino acids in length, one cantether a peptide to the amino terminus of β₂ microglobulin. If thesequence is appropriate, that peptide will bind to the MHC bindinggroove.

In other embodiments, a target cell is a naturally-occurring cell in abiological sample. Biological samples containing a target cell includegraft tissue; a biological sample which is being tested for the presenceof a target infected with an intracellular pathogen; a biological samplethat contains (or which is being tested for the presence of) a targetcell that is lysed by an autoimmune cell; a biological sample thatcontains (or which is being tested for the presence of) a target cellthat is lysed by a foreign (non-host) cell (e.g., a target of agraft-versus-host disease response); a biological sample that is beingtested for tissue compatibility with a prospective tissue/organrecipient; and the like.

Biological samples that are suitable for use depend on various factors,including the nature of the specific cell lysis being detected. Forexample, a blood sample may be used to determine histocompatibilitybetween potential donors and a prospective recipient. Blood samplesinclude whole blood samples, peripheral blood mononuclear cells, bloodcells enriched for particular sub-populations, etc.

Biological samples containing a naturally-occurring target cell includea cell in graft tissue or organ (e.g., tissue or organ that has beengrafted or is to be grafted into an individual), including, but notlimited to, bone marrow, skin, liver, pancreas, kidney, lung, heart,pancreatic islet cells, cord blood, whole blood, blood fractions, andthe like.

Biological sources of target cells also include a biological samplecontaining a cell that is infected with an intracellular pathogen,including, but not limited to, viruses (e.g., CMV HIV), bacteria (e.g.,Listeria, Mycobacteria, Salmonella (e.g., S. typhi) enteropathogenicEscherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC),Yersinia, Shigella, Chlamydia, Chlamydophila, Staphylococcus,Legionella), protozoa (e.g., Taxoplasma), fungi, and intracellularparasites (e.g., Plasmodium (e.g., P. vivax, P. falciparum, P. ovale,and P. malariae).

Labeling Target Cells

Target cells are labeled using any known method, including, e.g., themethod described in Example 1. Generally, cells are labeled in a mediumcontaining a buffer, and are labeled at room temperature (e.g., fromabout 19° C. to about 23° C.), although other temperatures, e.g.,physiological temperatures (e.g., 37° C.), can be used. Generally,incubation of the dye with the target cell for a period of from about 15seconds to about 2 minutes, although longer incubation periods may alsobe used. The dyes are readily taken up by the cells. Labeling with thecytosol-labeling fluorescent dye is stopped by addition of excessprotein, e.g., albumin, fetal bovine serum, and the like. One or morewashing steps can be performed after the cells are labeled. Labeledcells can be maintained at 4° C. for a period of time (e.g., 2 hours, 4hours, 8 hours, 12 hours, 24 hours, 48 hours, or longer) before beinganalyzed, if desired. Labeled cells can be further processed foranalysis, e.g., by flow cytometry. For example, labeled cells can betreated with 1% paraformaldehyde.

Detecting Cell Lysis

Target cell lysis is detected by detecting and/or measuringcytosol-labeling fluorescent dye remaining in the cytosol of the targetcell. Fluorescence is detected using a flow cytometry device or afluorescent microscope.

In some embodiments, target cell lysis is detected using a flowcytometry device. Flow cytometry devices and protocols are well known inthe art, and have been amply described in numerous publications. See,e.g., Flow Cytometry and Sorting, 2^(nd) ed. (1990) M. R. Melamed etal., eds. Wiley-Liss; Flow Cytometry and Cell Sorting, 2^(nd) ed. (2000)A. Radbruch, Springer-Verlag; and In Living Color: Protocols in FlowCytometry and Cell Sorting (2000) Diamond and Demaggio, eds,Springer-Verlag. Flow cytometry methods are also described in U.S. Pat.Nos. 5,968,738 and 5,804,387; the disclosures of which are hereinincorporated by reference. In certain embodiments, a flow cytometer thatis capable of multicolor analyses (e.g., 2, 4, 6, 8, or more differentcolors) is used.

Where fluorescence is detected using a flow cytometry device, lysis isdetected by detecting and/or measuring cytosol-labeling fluorescent dyeremaining in the cytosol of the cell. A reduction in the amount offluorescence indicates that cell lysis has occurred. For example, targetcell lysis can be expressed as 100%-% survival, where percentsurvival=(mean percent labeled cells/mean percent spontaneous release ofdye)×100.

Where the source of target cells is an organ or other solid tissue, andthe detection method is flow cytometry, the target cells are generallyare generally released from the organ or solid tissue using standardmethods, e.g. collagenase treatment.

Methods of Detecting Cells Having Lytic Activity

The present invention further provides methods of detecting the presenceof, or determining the number of, cells in a sample which have lyticactivity toward a target cell. A cell that has lytic activity toward atarget cell is referred to herein as an “effector cell.” The methodsgenerally involve labeling a target cell, as described above; contactingthe labeled target cell with a biological sample that is being testedfor the presence of a cell having lytic activity toward the target cell;and detecting specific lysis of the target cell. In many embodiments,the methods are performed in vitro.

Specific lysis of the target cell indicates the presence in the sampleof a lytic cell that is specific for the target cell. Whether lysis isspecific can be determined by including appropriate controls. Forexample, if the target cell is a cell line that is loaded with a peptideantigen, a control sample includes a cell of the same cell line notloaded with the peptide, or loaded with a different, non-cross-reactivepeptide.

In some embodiments, a biological sample is tested for the presence of acell having lytic activity toward an autologous cell, e.g., anautoreactive cytotoxic T lymphocyte (CTL). In these embodiments, targetcells include cells in a biological sample from an individual beingtested for the presence of autoreactive CTLs; and a cell loaded with apeptide known to be recognized by an autoreactive CTL. Target cells arelabeled as described above, then contacted with a biological sample fromthe individual. Specific lysis of the target cell indicates the presencein the sample from the individual of autoreactive CTLs. Control targetcells include a cell line not loaded with the peptide; or a biologicalsample from the individual that is known not to contain cells of thecell type recognized by a particular autoreactive CTL. For example,where the autoreactive CTL recognizes a pancreatic cell, a control cellis a cell from the same individual that is other than a pancreatic cell,e.g., a liver cell; and the like. These methods are useful to diagnosean autoimmune disorder that is due to the presence of autoreactive CTLs,including, but not limited to, Type 1 diabetes, autoimmune hepatitis,systemic lupus erythematosis; scleroderma; autoimmune thyroiditis;rheumatoid arthritis; myocarditis; and inflammatory bowel disease.

These methods are also useful in assessing the efficacy of a treatmentmethod for an autoimmune disorder that is due to the presence ofautoreactive CTLs. For example, a decrease in the number of autoreactiveCTLs following a particular treatment protocol indicates efficacy oftreatment.

In some embodiments, the methods provide for detection in a biologicalsample of a cell that mediates graft-versus-host disease (GVHD). GVHD iscaused by donor T-cells reacting against systemically distributedincompatible host antigens, causing powerful inflammation. In GVHD,mature donor T-cells that recognize differences between donor and hostbecome systemically activated. Frequently, the host isimmunocompromised, e.g., due to immunosuppressive chemotherapy, orcancer chemotherapy. In these embodiments, the biological sampleincludes graft tissue that is about to be engrafted into a host (e.g.,bone marrow cells); as well as graft tissue that has already beenintroduced into a host. Target cells are labeled as described above, andare generally host cells. These methods are useful for diagnosing GVHD,as well as for assessing the efficacy of a treatment for GVHD.

In other embodiments, the methods provide for detection in a biologicalsample of a cell that has lytic activity toward a tumor cell. In theseembodiments, the target cell is a tumor cell from an individual, and islabeled as described above. Cells from the same individual that havelytic activity toward the labeled tumor cells are detected by contactingthe labeled tumor cells with cells from the individual. The methods areuseful to assess the extent of a host CTL response to a tumor in thehost, as well as to assess the efficacy of a treatment protocol to boosta host's CTL response to a tumor.

The methods are also useful to detect the presence in an individual oftumor cells that are refractory to lysis by autologous CTLs. In theseembodiments, the labeled target tumor cells are contacted withautologous CTLs that specifically lyse the labeled tumor cells. Thepresence of labeled tumor cells that are not lysed by tumor-specificautologous CTLs indicates the presence in the individual of tumor cellsthat are refractory to lysis by autologous CTL. The presence of suchcell in the individual could suggest the need to modify a treatmentprotocol, e.g., to boost the host's immune response to such refractorycells.

In other embodiments, the methods provide for detection in a biologicalsample of a cell that has lytic activity toward a pathogen-infectedcell. In these embodiments, the target cell includes a cell line that isinfected in in vitro cell culture with the pathogen; a cell from anindividual that is infected in in vitro cell culture with the pathogen;and a cell from an individual that is known to be infected with a givenpathogen. The target cell is labeled, and contacted with a biologicalsample being tested for the presence of a cell that has lytic activitytoward a pathogen-infected cell. Lysis of the labeled target cellindicates the presence in the sample of such cells. Suitable controlsinclude target cells infected with an unrelated pathogen.

In other embodiments, the methods provide for detection ofantibody-dependent cell-mediated cytotoxicity (ADCC). In theseembodiments, target cells are typically pathogen-infected cells orcancerous cells. Lytic cells include, but are not limited to, NK cells,macrophages, eosinophils, monocytes, and neutrophils. A target cell islabeled as described above, and contacted with a sample comprisingantibody specific for the target cell; and a sample being tested for thepresence of lytic cells bearing on their cell surface an Fc receptor.Specific lysis of the target cell indicates the presence of such lyticcells. Suitable controls include labeled target cells not bound withspecific antibody, e.g., labeled target cells contacted with anon-specific antibody.

Methods of Detecting Antibody Having Lytic Activity

The invention also provides methods of detecting the presence in abiological sample of an antibody having lytic activity toward a targetcell. Biological samples to be tested for the presence of and/or anamount of, antibody having lytic activity toward a target cell includeserum. In some embodiments, components of the complement system areadded to the test sample, which comprises the labeled target cells andthe sample being tested for the presence of lytic antibody. Thebiological source of the target cells depends in part upon the nature ofthe lytic antibody being tested for. For example, where the lyticantibody is one that mediates lysis of red blood cells, sources oftarget cells include red blood cells.

Uses

The methods of the invention are useful in a variety of diagnostic andscreening methods. Diagnostic methods include methods of diagnosing adisease; methods for testing a treatment protocol that is beingdeveloped; and methods for testing the efficacy of a treatment protocol.Screening methods include methods of identifying an agent that increasesor decreases specific cell lysis. Screening methods are described inmore detail below.

The methods of the invention are useful in a variety of diagnosticapplications, e.g., in methods to determine tissue compatibility; inmethods to determine the presence and/or extent of graft-versus-hostreaction; in methods to detect the presence of and/or determine thenumber of CTL in an individual that have lytic activity toward anautologous tumor cell; in methods to detect the presence of and/ordetermine the number of tumor cells in a biological sample that are notlysed by autologous CTLs; in methods to detect the presence of and/ordetermine the number of autoreactive CTLs in a biological sample; inmethods to detect the presence of and/or determine the number of CTLs ina biological sample that are reactive toward a pathogen-infected cell;and in methods to detect the presence of and/or measure the amount of,antibody in a biological sample, which antibody has lytic activitytoward a cell.

In determining tissue compatibility (“tissue typing”) cells from a donorindividual (e.g., a potential donor) are mixed with cells taken from aprospective recipient and labeled. Sources of donor cells include, butare not limited to, autologous (self), xenogeneic sources, anHLA-matched sibling, an unrelated HLA-matched individual, a cadaver, orcloned tissue. Often, blood cells from a potential donor are mixed withblood cells from a recipient. Using the methods of the invention, thepresence of cells showing lytic activity toward recipient cells isdetected by detecting the amount of cytosol-labeling fluorescent dyeremaining in the labeled target population after mixing the labeledtarget population with the cells from the potential donor. Lack ofspecific lysis with a particular potential donor cell populationindicates the suitability of the donor as a source of tissues (e.g.,blood, organs, pancreatic cells, and the like) for that particularrecipient.

In determining the presence and/or extent of GVHD, cells from tissueengrafted into a host are removed from the host and mixed with hostcells. Host cells are labeled, as described above, and the presence ofcells showing lytic activity toward host cells is detected by detectingthe amount of cytosol-labeling fluorescent dye remaining in the labeledtarget population after mixing the labeled target population with thecells from the engrafted tissue.

In detecting the presence of and/or determining the number of CTLs in anindividual that have lytic activity toward an autologous tumor cell,cancerous cells are removed from the individual, labeled as describedabove, and mixed with CD8⁺ T lymphocytes from the individual. Thepresence of CTLs in the individual that have lytic activity toward thecancerous cells is detected by detecting the amount of cytosol-labelingfluorescent dye remaining in the labeled target tumor cell populationafter mixing the labeled target population with host CTLs. Such ananalysis can be conducted following treatment of the patient with acancer treatment protocol, to determine the efficacy of such treatment.Such an analysis is also useful to detect the presence in a populationof tumor cells of tumor cells that are refractory to lysis by apatient's CTLs. Where tumor cells that are refractory to lysis by apatient's CTLs are detected, a change in treatment protocol may beindicated.

The methods of the invention are also useful in vaccine development, andin testing the efficacy of a vaccine protocol. For example, a vaccinecomprising naturally occurring or synthetic components of a pathogenicorganism is tested. After administering the vaccine to an individual,the individual is tested for the number of pathogen-specific CTLs (e.g.,CTLs specific for one or more epitopes presented in the vaccine). Targetcells include cells of a cell line that have been loaded with syntheticor naturally occurring peptides from a pathogenic organism; or cellsfrom the individual that have been infected (e.g., in vitro) with thepathogen. Target cells are labeled as described above, and contacted invitro with a biological cell sample from the individual, e.g., a bloodsample. The presence of CTLs in the individual that have lytic activitytoward the target cells is detected by detecting the amount ofcytosol-labeling fluorescent dye remaining in the labeled target cellpopulation after mixing the labeled target cells with host cells.Suitable controls include cells of a cell line not loaded with peptide;and cells from the host that have not been infected in vitro with thepathogen.

The methods are also useful to evaluate an immune response (innate,humoral, and cellular) to an antigen. After an individual is exposed toan antigen (e.g., by immunization or by accidental exposure), the immuneresponse to the antigen can be determined by contacting a labeled targetcell, which displays the antigen (or an epitope of the antigen) on itscell surface, with cells (e.g., whole blood cells, PBMC's, asub-population of blood cells) from the individual, or with serum fromthe individual, and determining the extent of specific cell lysis. Insome embodiments, cells displaying the antigen or epitope on their cellsurface are a cell line loaded with peptide, such that the peptide isdisplayed in an MHC molecule. Such cells are useful to detect cellshaving specific lytic activity toward cells displaying the antigen.

Screening Methods

The present invention further provides methods of identifying agentsthat modulate specific lysis of a target cell. The methods generallyinvolve a sample that includes a labeled target cell and a lytic agentwith a test agent; and detecting fluorescence in the labeled targetcell. The target cell is labeled as described above with a fluorescentdye that labels the plasma membrane, and a fluorescent dye that labelsthe cytosol. As used herein, the term “modulate” encompasses “increase”and “decrease.”

A reduction in fluorescence in the labeled target cell, compared to acontrol sample, indicates that the agent increases specific cell lysis.Control samples do not contain the test agent. In some embodiments, asuitable control is a sample that includes a labeled target cell and alytic agent that effects specific lysis of the target cell. In thecontrol sample, the fluorescence decreases at a given rate. An increasein the rate of decrease of fluorescence in the sample that includes thetest agent indicates that the test agent increases specific cell lysis.In other embodiments, a suitable control is a sample that includes alabeled target cell and a lytic agent that does not effect lysis of thetarget cell, or effects lysis of the target cell inefficiently. In thecontrol sample, fluorescence does not decrease, or decreases at only avery low rate. An increase in the rate of decrease of fluorescence inthe sample that includes the test agent indicates that the test agentincreases specific cell lysis.

Agents of interest increase specific cell lysis by at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, or more, when compared with asuitable control. Agents that increase specific cell lysis are ofinterest in applications such as treating cancer, treating intracellularpathogen infections, and the like.

A decrease in reduction of fluorescence in the labeled target cell,compared to a control sample, indicates that the agent reduces specificcell lysis. Control samples do not contain the test agent. In someembodiments, a suitable control includes a labeled target cell and alytic cell that effects lysis of the target cell. In the control sample,fluorescence decreases at a given rate. A reduction in the rate ofdecrease of fluorescence in the sample that includes the test agentindicates that the test agent reduces specific cell lysis.

Agents of interest decrease specific cell lysis by at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, or more, when compared with asuitable control. Agents that decrease specific cell lysis are ofinterest in applications such as treating certain autoimmune disorders,GVHD, and the like.

The terms “test agent,” “candidate agent,” “substance,” and “compound”are used interchangeably herein. Candidate agents encompass numerouschemical classes, typically synthetic, semi-synthetic, ornaturally-occurring inorganic or organic molecules. Candidate agents maybe small organic compounds having a molecular weight of more than 50 andless than about 2,500 daltons. Candidate agents may comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, and may contain at least two of thefunctional chemical groups. The candidate agents may comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Agents further encompassinterfering RNA molecules, antibodies, and the like.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidation, etc. to producestructural analogs.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein—proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Thecomponents are added in any order that provides for the requisitebinding. Incubations are performed at any suitable temperature,typically between 4° C. and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening. Typically between 0.1 and 1 hour will besufficient.

Target cells are labeled as described above. In some embodiments, thelytic agent is an effector cell, as described above. In some of theseembodiments, the effector cell is not itself specific for a target cell;instead, specificity is imparted by an antibody specific for the targetcell, and the effector cell has an Fc receptor on its cell surface(e.g., a macrophage, neutrophil, monocyte, NK cell, and the like), asdescribed above. In other embodiments, the lytic agent is an antibody,as described above.

Typically, fluorescence is detected using flow cytometry. In someembodiments, the assays are adapted to a high through-put format.

Agents

The invention further provides agents identified using an assay methodof the invention. As discussed above, agents that increase specific celllysis are useful in treating cancer, in treating intracellular pathogeninfections, and the like. Agents that decrease specific cell lysis areuseful to treat GVHD, autoimmune disorders that involve specific celllysis, graft rejection, and the like.

Formulations

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredincrease or decrease in specific cell lysis.

The agent can be incorporated into a variety of formulations fortherapeutic administration. More particularly, the agents of the presentinvention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Other modes of administration will also find use with the subjectinvention. For instance, an agent of the invention can be formulated insuppositories and, in some cases, aerosol and intranasal compositions.For suppositories, the vehicle composition will include traditionalbinders and carriers such as, polyalkylene glycols, or triglycerides.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

An agent of the invention can be administered as injectables. Typically,injectable compositions are prepared as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of the agent adequate to achieve thedesired state in the subject being treated. Pharmaceutically acceptableexcipients have been amply described in a variety of publications,including, for example, A. Gennaro (2000) “Remington: The Science andPractice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins;Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Anselet al., eds 7^(th) ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed.Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Dosages

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is one that provides up to about 1 μgto about 1,000 μg or about 10,000 μg of an agent that increases ordecreases specific cell lysis can be administered in a single dose.Alternatively, a target dosage of an agent that increases or decreasesspecific cell lysis can be considered to be about in the range of about0.1-1000 μM, about 0.5-500 μM, about 1-100 μM, or about 5-50 μM in asample of host blood drawn within the first 24-48 hours afteradministration of the agent.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

Routes of Administration

An agent that increases or decreases specific cell lysis is administeredto an individual using any available method and route suitable for drugdelivery, including in vivo and ex vivo methods, as well as systemic andlocalized routes of administration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, intratumoral,subcutaneous, intradermal, topical application, intravenous, rectal,nasal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe agent and/or the desired effect. The composition can be administeredin a single dose or in multiple doses.

The agent can be administered to a host using any available conventionalmethods and routes suitable for delivery of conventional drugs,including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrastemal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of the agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

Kits with unit doses of the active agent, e.g. in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

Treatment Methods

The present invention further provides methods of treating disordersrelated to specific cell lysis. The methods generally involveadministering an effective amount of an active agent as described above.

Agents that decrease specific cell lysis are used to treat disordersassociated with specific cell lysis that is deleterious to a host,including, but not limited to, GVHD, and autoimmune disorders involvinglysis of “self” cells. An effective amount of an agent that decreasesspecific cell lysis is administered to an individual in need thereof. Aneffective amount is an amount that decreases specific cell lysis by atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or more, whencompared with specific cell lysis that occurs in the absence oftreatment with the active agent.

Individuals that are suitable for treatment include, but are not limitedto, individuals who have been diagnosed with GVHD; individuals at riskof developing GVHD (e.g., due to being a graft recipient); individualswho have been diagnosed with an autoimmune disorder that results fromdestruction of cells due to specific cell lysis (e.g., Type 1 diabetes);individuals who show symptoms of graft rejection; individuals who are atrisk of graft rejection; and the like.

Agents that increase specific cell lysis are used to treat disordersthat are amenable to treatment with specific cell lysis, e.g., where thehost has not mounted a CTL response or has mounted a CTL response thatis inadequate to effectively control the disorder. Such disordersinclude, but are not limited to, cancer, infection with an intracellularpathogen, and the like. An effective amount of an agent that increasesspecific cell lysis is administered to an individual in need thereof. Aneffective amount is an amount that increases specific cell lysis by atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or more, whencompared with specific cell lysis that occurs in the absence oftreatment with the active agent.

Kits

The present invention further provides kits for use in practicing one ormore of the above-described methods, where the subject kits include afluorescent dye for labeling the plasma membrane; a fluorescent dye forlabeling the cytosol. The dyes are present in a suitable storage medium,e.g., buffered solution, typically in a suitable container. In someembodiments, one or more antibodies specific for one or more cellsurface markers are included. In some embodiments, at least one of theantibodies is detectably labeled.

In some embodiments, a subject kit includes a cytosol-labelingfluorescent dye, a first plasma membrane-labeling fluorescent dye, andat least a second plasma membrane-labeling fluorescent dye, which secondplasma membrane-labeling fluorescent dye has spectral properties thatare distinguishable from the first plasma membrane-labeling fluorescentdye. In some of these embodiments, the cytosol-labeling fluorescent dyeis provided in a separate vial from the plasma membrane-labeling dyes.

In many embodiments, a plurality of plasma membrane-labeling dyes isprovided, each of which has spectral properties that are distinguishablefrom those of the other plasma membrane-labeling dyes. In otherembodiments, each of the plasma membrane-labeling dyes is providedpre-mixed separately with the cytosol-labeling dye.

In other embodiments, a subject kit a plasma membrane-labelingfluorescent dye, a first cytosol-labeling fluorescent dye, and at leasta second cytosol-labeling fluorescent dye, which second cytosol-labelingfluorescent dye has spectral properties that are distinguishable fromthe first cytosol-labeling fluorescent dye. In some of theseembodiments, the plasma membrane-labeling fluorescent dye is provided ina separate vial from the cytosol-labeling dyes. In many embodiments, aplurality of cytosol-labeling dyes is provided, each of which hasspectral properties that are distinguishable from those of the othercytosol-labeling dyes. In other embodiments, each of thecytosol-labeling dyes is provided pre-mixed separately with the plasmamembrane-labeling dye.

In other embodiments, a subject kit further includes target cells. Ingeneral, target cells are standard cell lines. Target cells aregenerally frozen. Target cells are stored in appropriate storage buffer,which may include one or more agents that preserve cell viability andreduce cell damage during thawing.

In still other embodiments, a subject kit further includes peptides thatcorrespond to a particular intracellular pathogen or tumor-associatedantigen.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, compact disk, etc., on which the information has beenrecorded. Yet another means that may be present is a website addresswhich may be used via the internet to access the information at aremoved site. Any convenient means may be present in the kits.

Systems

The present invention further provides a system for detecting specificcell lysis. A subject system generally includes a plasma-labelingfluorescent dye; a cytosol-labeling fluorescent dye; a target cell; anda device for detecting fluorescence in a cell, e.g., a flow cytometer. Asubject system may further include a lytic agent. A subject system mayfurther include one or more additional dyes or other detectable markers,as described above. A subject system is used to perform a diagnostic ora screening method described herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Antigen-Specific Lysis of Target Cells Labeled with PKH-26 andCFSE

This example describes an assay termed “fluorometric assessment of Tlymphocyte antigen-specific lysis” or FATAL, and compares this assay tothe ⁵¹Cr release assay.

Materials and Methods

Generation of Effector Cells

To evaluate antigen specific CD8⁺ T-cell effector activity,cytomegalovirus (CMV) specific CTLs were used as effector cells andB-lymphoblastoid cell lines (BCL) as target cells. Several CMV-pp65peptide (495-503: NLVPMVATV; SEQ ID NO:1) specific semi-clones from anHLA-A*0201, CMV seropositive donor were generated as previouslydescribed (Wills et al. (1996) J. Virol. 70:7569-7579).

Preparation of Target Cell Populations

BCL were used as target cells in both the ⁵¹Cr release and FATAL assays.Both assays were performed with immortalized BCL from an HLA-A*0201individual. The BCL were incubated with 10 μg/ml of the HLA-A*0201restricted cytomegalovirus (CMV)-pp65 (495-503: NLVPMVATV; SEQ ID NO:1)peptide for 1 hour at 37° C. in 5% CO₂ prior to use in the assays. AnHLA-A*0201 restricted HIV-Gag peptide (77-85: SLYNTVATL; SEQ ID NO:2)was used as a negative control. In addition, target BCL were incubatedwithout peptide as background controls. Further experiments used BCLinfected with recombinant vaccinia virus (rVV) overnight expressing CMVantigen (CMVpp65-rVV) or control vaccinia (Vaccinia Thymidine kinasenegative, TK) (Therion Biologics, Cambridge, Mass.). To confirmHLA-A*0201 restricted specific lysis, HLA mismatched BCL were includedin the assays. All target cells were washed once in PBS, centrifuged for5 minutes at 400%g, the supernatant was discarded and cells wereresuspended prior to use. (Biowhittaker, Walkersville, Md.). The cellnumber and viability were assessed by trypan blue exclusion(Biowhittaker). The antigen labeled target and control cells weresubsequently used in the FATAL and ⁵¹Cr release cytotoxicity assays asdescribed below. In order to compare the ⁵¹Cr release and FATAL assays,all experiments were performed in parallel.

¹⁵Cr Release Assay

A standard ⁵¹Cr release assay was performed as previously described (Ogget al., 1998). Peptide pulsed, CMV pp65-rVV infected and control targetsfor use in the ⁵¹Cr release assay were labeled with 100 μCi ⁵¹Cr assodium chromate (Na₂ ⁵ CrO₄), (New England Nuclear, Boston, Mass.) andincubated for 1 h (37° C., 5% CO₂). Target cells were washed three timesin R-15 medium (RPM1 1640 media supplemented with 15% heat inactivatedfetal calf serum, L-glutamine, HPEPS, and penicillin-streptomycin(Biowhittaker)) and aliquoted in duplicate at 5×10³ cells per well into96-well U-bottom plates (Becton Dickinson Labware, Franklin Lakes,N.J.). To compare the relative sensitivity of the ⁵¹Cr and FATAL assays,CMV-pp65 specific CD8⁺ T cells were incubated at various ratios withtarget cells. The assay was incubated at 37° C., 5% CO₂ for up to 5hours. After the assay incubation period, 30 μl of the supernatant washarvested from each well into corresponding wells of a 96-well LumaPlate(Packard Instruments, Downers Grove, Ill.). The gamma irradiation fromeach well was assessed in a TopCount Microplate Scintillation Counter(Packard Instruments). For the ⁵¹Cr release assays, the percent specificlysis was calculated by the following equation:

Percent specific LYSIS=(test release−spontaneous release/maximumrelease−spontaneous release)×100.

All the assays were performed in duplicate.

Flow Cytometric Assay

Antigen labeled and control target cells for use in the FATAL assay werelabeled with PKH-26 according to the manufacturer's instructions (Sigma,St. Louis, Mo.). 1×10⁶ target cells were stained with PKH-26 (finalconcentration of 2.5×10⁻⁶ M) at room temperature for 3-5 min. To stopthe reaction a volume of heat inactivated fetal calf serum (FCS) equalto that of the cells and dye, was added to the cell suspension, thenincubated for 1 min at room temperature before centrifugation for 5minutes at 400× g. After a single wash with 10 ml PBS, the target cellswere centrifuged for 5 minutes and the supernatant was discarded. PKH-26labeled target cells were then labeled with 5-(and-6)-carboxyfluoresceindiacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, Oreg.).CFSE was diluted to a final concentration of 2.5×10⁻⁶ M and added to thetarget cells suspension immediately following the addition of the CFSE,an equal volume of FCS was added to stop the reaction and cells werecentrifuged for 5 minutes at room temperature. Finally, the target cellswere washed twice with PBS, resuspended in R-15 medium, and dispensed induplicate at 5×10³ cells per well into 96-well U-bottom plates (BectonDickinson). Effector cells were added at various E:T ratios and mixedwith the target cells.

The FATAL assay was incubated up to 5 hours at 37° C., 5% CO₂ inparallel with the ⁵¹Cr release assay. Following incubation, the totalcontents of the U bottom plate were transferred to a 96-well V-bottomplate (Becton Dickinson, Lincoln Park, N.J.) and centrifuged for 5minutes at room temperature. The supernatant was discarded and the cellpellet was resuspended in 150 μl 1% paraformaldehyde and analyzed byflow cytometry within 24 hours.

Flow Cytometry

Flow cytometry was performed with a FACS Calibur (Becton Dickinson, SanJose, Calif.) equipped with an argon laser operating at 488 nm.Fluorescence was collected through a 530/25 nm filter for CFSE emissionand through a 585/40 nm filter for PKH-26. The contents of each FATALassay well (150 μl) were acquired and no gating was used foracquisition. A threshold was set on forward light scatter and side lightscatter to exclude a group of very small scatter signals at the lowerleft corner of the plot, previously characterized as subcellularfragments (Bartkowiak et al., 1999). Data was subsequently analyzed viaCellQuest software (Becton Dickinson).

Results

Labeling with PKH-26 and CFSE is Uniform and Does not Affect CellViability.

To determine the efficiency of labeling, BCL were stained withPKH-26/CFSE in combination and then analyzed by flow cytometry. Thestaining of BCL target cells is shown in FIG. 1, with quadrant positionsand instrument settings that were defined by unstained target cells.FIG. 1a shows a forward scatter/side scatter (FSC/SSC) dot plot and anR1 gate determined by target cells incubated in media alone. BCL werestained with either PKH-26 or CFSE alone (FIG. 1b) and the resultinghistogram plots are shown in FIG. 1c. Color compensations were set usingstandard procedures (Loken and Lanier, 1984). The analysis markers wereadjusted to exclude autofluorescence found to the left of the cursorsdefining the target population.

Target Cell Labeling and Calculation of Specific Cell Lysis as Detectedby the FATAL Assay.

PKH-26/CFSE double stained BCL target cells were co-cultured withnon-stained effector cells. FIG. 1c shows histogram displays of BCLautofluorescence (FIG. 1c(i,iv)), the CFSE and PKH-26 fluorescence ofcontrol targets alone (FIG. 1c(ii,v)) and targets co-incubated witheffector cells (FIG. 1c(iii,vi)). Histograms FIG. 1c(iii) and FIG.1c(vi) show two exclusive peaks, confirming the clear identification oftwo separate populations. There was no change in the mean position ofthe two peaks in the co-cultured sample from the single stained controlsindicating that there was no transfer of PKH-26 or CFSE to the effectorcell population. The assessment of antigen specific cell lysis asdetected by the FATAL assay was determined by the percentage of labeledtarget cells surviving following a 4-hour incubation with effectorcells. Target cells incubated in the absence of effector cells(spontaneous release of CFSE) were used for comparative analysis.Initially, a forward scatter/side scatter (FSC/SSC) dot plot was drawn(FIG. 1a), and live gate (R1) was adjusted to include viable cells.

This determination is made by the analysis of target cells incubatedalone with media in the assay plate. From the R1 live gate a PKH-26histogram (FIG. 1c(iii)) is derived, which distinguishes the target (R2)and effector (R3) cell populations. The discrimination of target cellsfrom effector cells permits the calculation of an actual E:T ratio foreach condition (FIG. 1c(iii)). A second histogram displays the CFSEfluorescence (FIG. 2) of the target cells identified by the PKH-26^(Hi)gate (R2). Cells contained within the PKH-26^(Hi) and CFSE^(Hi) doublepositive gate represent viable target cells. A marker (M1) was set usingcontrol conditions so that the CFSEHi viable target cell populationincluded >98% of the target cells incubated in media alone. Thepercentage of specific cell lysis in the viable target cell populationwas determined by the disappearance of the antigen labeled targets fromCFSE^(Hi) population when compared to the CFSE^(Hi) fluorescence ofcontrol targets. The position of the PKH-26^(Hi) and CFSE^(Hi) gates wasmaintained throughout the analysis.

Therefore, to calculate the percentage of specific lysis using the FATALassay (FIG. 2): Percent SURVIVAL=(mean CFSE^(Hi) percent of testwell/mean CFSE^(Hi) percent of spontaneous release)×100. Percentspecific LYSIS=100-% survival.

PKH-26/CFSE Staining is Retained Significantly Longer than ⁵¹ CrLabeling.

To compare the leakage of ⁵¹Cr and PKH-26/CFSE, BCL were labeled andspontaneous release monitored over a 45-h period. Labeled target cellswere aliquoted at 1×10⁴ cells per well and the results are graphicallyrepresented in FIG. 3. The overall leakage of PKH-26 and CFSE from cellsincubated at 37° C., 5% CO₂ was forty fold lower than that found for⁵¹Cr following a 45-hour incubation. Spontaneous release of ⁵¹Cr over30% is sufficient to invalidate an assay, limiting ⁵¹Cr release assaysto less than 18 hours. Following the 45-hour incubation, the leakage ofPKU-26/CFSE was less than 5%, indicating that PKH26 and CFSE are viablecomponents of a short or long term cytolysis assay.

Comparison of Specific Target Cell Lysis Between the FATAL and ⁵¹CrRelease Assays

A direct comparison of cytotoxic activity measured by the FATAL and ⁵¹Crrelease assays was performed concurrently using identical antigenspecific effector, target cells and E:T ratios. Both the FATAL and ⁵¹Crrelease assay results are displayed in FIG. 4. These data represent themean of duplicate samples. It can be seen that at high E:T ratios thepercentage of lysed target cells in the ⁵¹Cr release assay is higherthan that seen using the FATAL assay. Importantly, at lower E:T ratiosthe FATAL assay detected a greater degree of viable target cell lysis.Regression analysis demonstrated a significant correlation between thelysis detected by the two assays (r2=0.998, P<0.0001).

Additionally, target cells were infected with rVV overnight andsubsequently labeled with PKH-26/CFSE or ⁵¹Cr prior to incubation withantigen specific effector cells. Analysis of specific cell lysisdetected by FATAL or ⁵¹Cr indicated that the techniques were comparable.

Example 2 Antigen-Specific Lysis of Dual Target Cells Labeled withPKH-26 or DiD and CFSE.

This example describes an assay termed “Dual Target assay”.

Materials and Methods

Generation of Effector Cells

To evaluate antigen specific CD8⁺ T-cell effector activity, Influenzamatrix protein-specific CTLs were used as effector cells andB-lymphoblastoid cell lines (BCL) as target cells. Several influenza HLAA*0201 peptide (GILGFVFTL SEQ ID NO:3) specific semi-clones from anHLA-A*0201, donor were generated as previously described (Wills et al.(1996) J. Virol. 70:7569-7579).

Preparation of Target Cell Populations

The dual target assay was performed using the same autologousimmortalized BCL from an HLA-A*0201 individual. The BCL were incubatedwith 10 μg/ml of the HLA-A*0201 restricted influenza HLA A*0201 peptide(GILGFVFTL SEQ ID NO:3) peptide for 1 hour at 37° C. in 5% CO₂ prior touse in the assays. In addition, target BCL were incubated withoutpeptide as background inter- and intra-assay controls. All target cellswere washed once in PBS, centrifuged for 5 minutes at 400 g, thesupernatant was discarded and cells were resuspended prior to use.(Biowhittaker, Walkersville, Md.). The cell number and viability wereassessed by trypan blue exclusion (Biowhittaker). The antigen labeledtarget and control cells were subsequently used in the Dual Target Assayas described below.

Flow Cytometric Assay

Peptide labeled and control target cells for use in the FATAL assay werelabeled with PKH-26 or DiD(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate)according to the manufacturer's instructions (Sigma, St. Louis, Mo. andMolecular Probes, Eugene, Oreg. respectively). 1×10⁶ target cells werestained with PKH-26 (final concentration of 2.5×10⁻⁶ M) at roomtemperature for 3-5 minutes. To stop the reaction, a volume ofheat-inactivated fetal calf serum (FCS) equal to that of the cells anddye was added to the cell suspension, then incubated for 1 minute atroom temperature before centrifugation for 5 minutes at 400× g. After asingle wash with 10 ml PBS, the target cells were centrifuged for 5minutes and the supernatant was discarded. 1×10⁶ target cells werestained with DiD (final concentration of 1×10⁻⁶ M) at room temperaturefor 5-10 min. To stop the reaction, a volume of heat-inactivated fetalcalf serum (FCS) equal to that of the cells and dye was added to thecell suspension, then incubated for 1 minute at room temperature beforecentrifugation for 5 minutes at 400× g. After a single wash with 10 mlPBS, the target cells were centrifuged for 5 minutes and the supernatantwas discarded.

DiD or PKH-26 labeled target cells were then labeled with5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE)(Molecular Probes, Eugene, Oreg.). CFSE was diluted to a finalconcentration of 2.5×10⁻⁶ M and added to the target cells suspension.Immediately following the addition of the CFSE, an equal volume of FCSwas added to stop the reaction and cells were centrifuged for 5 minutesat room temperature. Finally, the target cells were washed twice withPBS, resuspended in R-15 medium, and dispensed in duplicate at 5×10³cells per well into 96-well U-bottom plates (Becton Dickinson). Effectorcells were added at various E:T ratios and mixed with the target cells.

The Dual target assay was incubated up to 5 hours at 37° C., 5% CO₂.Following incubation, the total contents of the U bottom plate weretransferred to a 96-well V-bottom plate (Becton Dickinson, Lincoln Park,N.J.) and centrifuged for 5 minutes at room temperature. The supernatantwas discarded and the cell pellet was resuspended in 100 μl 1%paraformaldehyde and analyzed by flow cytometry within 24 hours.

Flow Cytometry

Flow cytometry was performed with a FACS Calibur (Becton Dickinson, SanJose, Calif.) equipped with an argon laser operating at 488 nm.Fluorescence was collected through a 530/25 nm filter for CFSE emission,through a 585/40 nm filter for PKH-26 and a 661/16 filter for DiD. Thecontents of each Dual Target Assay well (100 μl) were acquired and nogating was used for acquisition. A threshold was set on forward lightscatter and side light scatter to exclude a group of very small scattersignals at the lower left corner of the plot, previously characterizedas subcellular fragments (Bartkowiak et al., 1999). Data weresubsequently analyzed via CellQuest software (Becton Dickinson).

Results

The results are shown in FIGS. 5-8. FIGS. 5A and 5B depict FACS plotsshowing staining of donor BCL with CFSE and either PKH-26 (FIG. 5A) orDiD (FIG. 5B), and analysis of each stained cell populationindividually. Donor BCL were stained with CFSE and either PKH-26 (A) orDiD (B) and incubated for 4 hours at 37 C independently. Regions R1 andR2 are gated on PKH-26 and DiD respectively. The percentage of CFSEpresent in the double stained populations was obtained by drawing amarker region (M1) on the CFSEHi population.

FIG. 6 presents results showing that dual-labeled target cells arereadily distinguishable in mixed populations using flow cytometry. DonorBCL were stained with CFSE and either PKH-26 or DiD. The two sets oftargets were then co-incubated for 4 hours at 37 C. The percentage ofCFSE present in the double stained populations was obtained by drawing amarker region (M1) on the CFSE^(Hi) population. FIG. 6 depicts the CFSEhistogram gated on PKH-26^(Hi) (upper right panel) or gated on DiD^(Hi)(lower right panel). Here we demonstrate that the signal of both dyes inthe same culture can be distinguished by the appearance in FL-2 and FL-4for PKH-26 and DiD, respectively.

FIG. 7 shows the results of contacting unpulsed labeled target cellswith a lytic agent. Donor BCL were stained with CFSE and either PKH-26or DiD. The two sets of targets were then co-incubated for 4 hours at 37C in the presence of autologous influenza matrix HLA-A*0201 peptidespecific T-cells. Regions R1 and R2 are gated on PKH-26 and DiD,respectively. The percentage of CFSE present in the double stainedpopulations was obtained by drawing a marker region (M1) on theCFSE^(Hi) population. This “unpulsed” coculture represents thebackground lysis of targets in the absence of specific Influenza matrixHLA-A*0201 peptide lysis.

FIG. 8 shows the results of contacting peptide-pulsed labeled targetcells with a lytic agent. Two populations of donor BCL were incubatedwith or influenza matrix HLA-A*0201 peptide for 1 hour (pulsed). Thepeptide pulsed BCL were stained with CFSE and PKH-26, whereas theunpulsed BCL were stained with CFSE and DiD. The two sets of targetswere then co-incubated (E) for 4 hours at 37 C in the presence ofautologous influenza matrix HLA-A*0201 peptide specific T-cells.Following the incubation period, the percentage of labeled BCL wascalculated for each stained population by comparison with unpulsed BCLincubated with effector T-cells (D). The percentage survival for each ofthe targets in the dual target assay was calculated using the equation:(% of CFSE^(Hi) (M1) of the PKH-26 or DiD costained populations (E)/% ofCFSE^(Hi) (M1) of the PKH-26 or DiD costained populations (D))×100. Thepercentage lysis is obtained by subtracting the percentage survival from100. The example given here cocultures D and E is: (3.83/9.57)×100=40%survival. Therefore 60% of the target cells labeled with PKH-26/CFSEwere lysed.

The above demonstrate that the invention provide methods for detectingspecific cell lysis. These methods involve the labeling methods thateliminate the use of radioactivity, as required in previous ⁵¹Cr releaseassays, and provide the advantages of uniform labeling of virtually allcell types to produce a bright and distinct labeled target cellpopulation which is easily identified by flow cytometry. Lysis of thelabeled target population using the methods of the present invention isreadily detected, as the cytosolic label has a very low rate ofspontaneous leakage, and, when membrane damage occurs, the dye is almostinstantaneously lost and the cells are no longer able to take up orretain the dye. These assay methods provide detailed information aboutboth target and effector cells at a single cell level, and are moresensitive that ⁵¹Cr release assays.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 3 <210> SEQ ID NO 1 <211> LENGTH: 9<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic peptide <400> SEQUENCE: 1Asn Leu Val Pro Met Val Ala Thr Val  1               5 <210> SEQ ID NO 2<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: synthetic peptide<400> SEQUENCE: 2 Ser Leu Tyr Asn Thr Val Ala Thr Leu  1               5<210> SEQ ID NO 3 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic peptide <400> SEQUENCE: 3Gly Ile Leu Gly Phe Val Phe Thr Leu  1               5

What is claimed is:
 1. A method of detecting specific lysis of a targetcell, comprising: a) contacting a labeled viable target cell with alytic agent, wherein the target cell is labeled with a first plasmamembrane-labeling fluorescent dye that labels the plasma membrane of thetarget cell and a first cytosol-labeling fluorescent dye that labels thecytosol of the target cell; and b) determining the amount of thecytosol-labeling fluorescent dye remaining in the target cell, wherein areduction in the amount of the cytosol-labeling fluorescent dye in thetarget cell when the target cell is contacted with the lytic agent,compared to the amount of cytosol-labeling fluorescent dye in a controltarget cell not contacted with the lytic agent, indicates that thetarget cell is lysed by the lytic agent, and wherein said first plasmamembrane-labeling fluorescent dye has detectably different spectralproperties from the first cytosol-labeling fluorescent dye such that thefirst plasma membrane-labeling fluorescent dye is distinguishable fromthe first cytosol-labeling fluorescent dye.
 2. The method of claim 1,wherein said first plasma membrane-labeling fluorescent dye is alipid-associated fluorescent dye, and wherein said firstcytosol-labeling fluorescent dye is a fluorescent dye that labelsproteins in the cytosol.
 3. The method of claim 1, wherein the lyticagent is a cell having lytic activity toward the target cell.
 4. Themethod of claim 3, wherein the cell having lytic activity toward thetarget cell is an antigen-specific CD8⁺ T lymphocyte, and the targetcell displays the antigen in an MHC Class I molecule on its cellsurface.
 5. The method of claim 1, wherein the lytic agent is anantibody specific for a cell surface marker on the target cell.
 6. Themethod of claim 1, wherein the lytic agent comprises an antibodyspecific for a cell surface marker on the target cell, and a lytic cellhaving an Fc receptor, wherein the lytic cell is selected from the groupconsisting of a neutrophil, an eosinophil, a macrophage, a monocyte, anda natural killer cell.
 7. The method of claim 1, wherein the emission ofthe frst plasma membrane-labeling fluorescent dye differs from theemission of the first cytosol-labeling fluorescent dye by at least about10 nm.
 8. The method of claim 2, wherein the lipid-associatedfluorescent dye is selected from PKH-26, PKH-67, and a long chaindialkylcarbocyanine.
 9. The method of claim 2, wherein theprotein-labeling cytosol dye is selected from 5-(-6)-carboxyfluorescein,5-(-6)(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine),7-amino-4-chloromethylcoumarin, and a SNARF® fluorescent dye.
 10. Themethod of claim 1, wherein the amount of cytosol-labeling fluorescentdye in the target cell is determined using flow cytometry.